Multiple drug resistance refers to acquisition of broad range of resistance phenotypes through genetic changes at a small number of loci. Multidrug resistance is a clinical problem in chemotherapeutic treatment of tumors and infectious disease. We are studying pleiotropic drug resistance (Pdr) in Saccharomyces cerevisiae as a model for eukaryotic multiple drug resistance. Expression of an ATP-binding cassette transporter protein, Pdr5p, is critical in multidrug resistance of S. cerevisiae. We have recently discovered a new signaling pathway causing particular mitochondrial mutants lacking the Fo subcomplex of the ATPsynthase to activate function of the transcription factor Pdr3p, that in turn stimulates PDR5 expression. The goal of this proposal is to identify the signal and participants in the pathway leading to activation of nuclear Pdr3p. Preliminary experiments indicate that the level of Fo subunits is a key modulator of Pdr3p activity. To directly explore this relationship, we will use Fo mutants and overexpression vectors to assess the link between Fo components and Pdr3p activity. Genetic analysis will be employed to identify negative regulators of Pdr3p. A library of gene disruption mutations will be screened for null mutations that activate Pdr3p while a chemical mutagenesis approach will be undertaken to identify essential genes and gain-of function alleles that can stimulate Pdr3p function. We have identified the novel protein Yp1055cp as a participant in the mitochondrial-nuclear (retrograde) pathway controlling Pdr3p. Two hybrid and co-immunoprecipitation experiments will be used to determine if these two proteins directly interact. A protein (Lsm1p) involved in mRNA degradation is also required for normal retrograde signaling to Pdr3p. The role of Lsm1p and Yp1055cp in control of Pdr3p will be investigated by genetic analysis. We will directly screen for other participants in Pdr3p retrograde signaling by curing the disruption library of mitochondrial genomic DNA (rho0) which will result in constitutive Pdr3p activation. The resulting mutants will be tested for mutants that reduce cycloheximide resistance. Strikingly, a similar phenomenon in which ABC transporter genes are up-regulated in rho0 cells has been described in the pathogenic yeast Candida glabrata. Our study of this phenomenon in the genetically-tractable S. cerevisiae cell will allow us to more rapidly understand this important facet of multidrug resistance.